Challenges in tissue section mass spectrometry imaging: How to select a high-sensitivity mass spectrometry imaging source

Tissue sections are the most common yet most challenging sample type for mass spectrometry imaging. Sections are thin and have intricate structures, with molecular distribution variations occurring even at the micrometre scale; if the imaging source lacks sufficient sensitivity, faint signals are drowned out by noise, resulting in an ‘unclear’ image. This article addresses the challenges of section imaging and explains the rationale behind selecting a high-sensitivity imaging source.

Three typical challenges in section imaging

Firstly, there is the matrix background. Traditional MALDI methods require the application of a matrix onto the section; the small molecules in the matrix themselves form dense peaks at the low-mass end, suppressing the signal from target molecules, and can also introduce spatial artefacts due to uneven application. The second is insufficient sensitivity. Many endogenous metabolites and lipids are naturally present in low concentrations within tissues; when conventional ionisation efficiency is low, the signal-to-noise ratio of the imaging map is poor. The third is narrow polarity coverage. Certain non-polar species, such as lipids and steroids, are difficult to capture using a single ionisation method, resulting in ‘gaps’ in the imaging map.

Where does sensitivity come from: the gain from post-desorption photoionisation

A direct approach to enhancing sensitivity is to introduce a post-photoionisation (secondary photoionisation) step following desorption. The DESI/PI dual-mode of New Ceyuan’s MSI DPI involves subjecting the vapour-phase molecules desorbed by DESI to a further soft ionisation via photochemical ionisation before they enter the mass analyser. This step boosts the ionisation efficiency of weakly polar and non-polar species, enhancing the overall signal by approximately 1–3 orders of magnitude.

In validated samples, the gain is quantifiable: theanine was enhanced by approximately 14.5-fold, caffeine by approximately 61-fold (approximately 60-fold), and cholesterol in mouse brain by approximately 205-fold. These figures reflect the actual incremental gain provided by the dual-mode coverage and are key to making faint lipids and metabolites in sections ‘visible’.

How does resolution match the scale of the section?

The resolution requirements for section imaging depend on the research question. MSI DPI offers spatial resolution of 20–200 μm, making it suitable for visualising areal distributions such as tissue regions, brain areas and tumour boundaries. If the focus is on molecular gradients at the subcellular level (such as concentration changes near cell junctions or nerve endings), the 2–3 μm resolution of MSI LDPI is more appropriate; with a minimum detectable m/z of approximately 70, it can characterise distributions at a finer scale.

Matrix-free imaging is gentler on sections

For tissue sections, eliminating matrix spraying is not merely a matter of streamlining the process; it is crucial for data quality. Without matrix background, signals from small molecules at the low-mass end are cleaner; without uneven spraying, spatial registration is more reliable. Both MSI DPI and LDPI are matrix-free, which is particularly important for precious, irreplaceable clinical sections—the samples are not damaged by chemical matrices and can still undergo combined analyses such as HE staining.

Selection Checklist: Matching Pain Points to Source Characteristics

When considering section imaging, we recommend checking against this list: Does the sample contain large amounts of non-polar species such as lipids (if so, prioritise a source with post-electron ionisation); is the spatial resolution regional or cellular (determining whether to use DPI or LDPI); whether the samples are precious and must not be damaged (in which case, a matrix-free approach should be prioritised); and whether there is an existing host system available for retrofitting (which determines whether to procure an imaging source rather than a complete instrument). By matching each challenge to the source’s characteristics item by item, the selection process will remain on track.

Summary

The challenges in tissue section mass spectrometry imaging lie in sensitivity, background noise and polarity coverage. The key to a high-sensitivity imaging source lies in using post-electron ionisation to amplify faint, non-polar signals, employing matrix-free analysis to suppress background noise, and utilising gradient resolution to accommodate different scales. NewSource’s MSI DPI / LDPI integrates these three elements into a single retrofit solution, elevating tissue section imaging from merely ‘being able to image’ to ‘imaging with clarity’.